Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii).

Peyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology-guided gene discovery strategy to elucidate a near-complete biosynthetic pathway from l-tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3-hydroxylation of l-tyrosine to l-DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate-specific and regiospecific substituted phenethylamine O-methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N-methyltransferase displaying broad substrate specificity and leading to the production of N-methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation.

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Compartmentalization at the interface of primary and alkaloid metabolism.

Plants produce many compounds used by humans as medicines, including alkaloids of the benzylisoquinoline (BIA), monoterpene indole (MIA) and tropane classes. The biosynthetic pathways of these pharmaceutical alkaloids are complex and spatially segregated across several tissues, cell-types and organelles. This review discusses the origin of primary metabolic inputs required by these specialized biosynthetic pathways and considers aspects relevant to their spatial organization. These factors are important for alkaloid production both in the native plants and for synthetic biology pathway reconstruction in microorganisms.

Back to the plant: overcoming roadblocks to the microbial production of pharmaceutically important plant natural products.

Microbial fermentation platforms offer a cost-effective and sustainable alternative to plant cultivation and chemical synthesis for the production of many plant-derived pharmaceuticals. Plant alkaloids, particularly benzylisoquinoline alkaloids and monoterpene indole alkaloids, and recently cannabinoids have become attractive targets for microbial biosynthesis owing to their medicinal importance. Recent advances in the discovery of pathway components, together with the application of synthetic biology tools, have facilitated the assembly of plant alkaloid and cannabinoid pathways in the microbial hosts Escherichia coli and Saccharomyces cerevisiae. This review highlights key aspects of these pathways in the framework of overcoming bottlenecks in microbial production to further improve end-product titers. We discuss the opportunities that emerge from a better understanding of the pathway components by further study of the plant, and strategies for generation of new and advanced medicinal compounds.

Prospects for Carotenoid Biofortification Targeting Retention and Catabolism.

Due to the ongoing prevalence of vitamin A deficiency (VAD) in developing countries there has been a large effort towards increasing the carotenoid content of staple foods via biofortification. Common strategies used for carotenoid biofortification include altering flux through the biosynthesis pathway to direct synthesis to a specific product, generally ß-carotene, or via increasing the expression of genes early in the carotenoid biosynthesis pathway. Recently, carotenoid biofortification strategies are turning towards increasing the retention of carotenoids in plant tissues either via altering sequestration within the cell or via downregulating enzymes known to cause degradation of carotenoids. To date, little attention has focused on increasing the stability of carotenoids, which may be a promising method of increasing carotenoid content in staple foods.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.